Liposomal Binuclear Ir(III)–Cu(II) Coordination Compounds with Phosphino-Fluoroquinolone Conjugates for Human Prostate Carcinoma Treatment

Novel heteronuclear IrIII–CuII coordination compounds ([Ir(η5-Cp*)Cl2Pcfx-Cu(phen)](NO3)·1.75(CH3OH)·0.75(H2O) (1), [Ir(η5-Cp*)Cl2Pnfx-Cu(phen)](NO3)·1.75(CH3OH)·0.75(H2O) (2), [Ir(η5-Cp*)Cl2Plfx-Cu(phen)](NO3)·1.3(H2O)·1.95(CH3OH) (3), [Ir(η5-Cp*)Cl2Psfx-Cu(phen)] (4)) bearing phosphines derived from fluoroquinolones, namely, sparfloxacin (Hsfx), ciprofloxacin (Hcfx), lomefloxacin (Hlfx), and norfloxacin (Hnfx), have been synthesized and studied as possible anticancer chemotherapeutics. All compounds have been characterized by electrospray ionization mass spectrometry (ESI-MS), a number of spectroscopic methods (i.e., IR, fluorescence, and electron paramagnetic resonance (EPR)), cyclic voltammetry, variable-temperature magnetic susceptibility measurements, and X-ray diffractometry. The coordination geometry of IrIII in all complexes adopts a characteristic piano-stool geometry with the η5-coordinated and three additional sites occupied by two chloride and phosphine ligands, while CuII ions in complexes 1 and 2 form a distorted square-pyramidal coordination geometry, and in complex 3, the coordination geometry around CuII ions is a distorted octahedron. Interestingly, the crystal structure of [Ir(η5-Cp*)Cl2Plfx-Cu(phen)] features the one-dimensional (1D) metal–organic polymer. Liposomes loaded with redox-active and fluorescent [Ir(η5-Cp*)Cl2Pcfx-Cu(phen)] (1L) have been prepared to increase water solubility and minimize serious systemic side effects. It has been proven, by confocal microscopy and an inductively coupled plasma mass spectrometry (ICP-MS) analysis, that the liposomal form of compound 1 can be effectively accumulated inside human lung adenocarcinoma and human prostate carcinoma cells with selective localization in nuclei. A cytometric analysis showed dominance of apoptosis over the other cell death types. Furthermore, the investigated nanoformulations induced changes in the cell cycle, leading to S phase arrest in a dose-dependent manner. Importantly, in vitro anticancer action on three-dimensional (3D) multicellular tumor spheroids has been demonstrated.


EPR measurements
Figure S12 The X-band EPR spectra of 1 -4 at 77 K together with the spectrum calculated by computer simulation of the experimental spectra with spin Hamiltonian parameters given in the text……………………………………….S32 Figure S13 EPR frozen solution spectra (at 77 K) of compounds 1 -4; in DMSO solvent together with the theoretical spectrum calculated using the parameters given in the text……………S33   Figure

Figure S4
Packing diagram of complex IrPLmCu S10 Additionally, the binuclear unit is stabilized by π-stacking interactions between phenanthroline and lomefloxacin fragments.

Figure S5
Packing diagram of complex 1 showing (A) π-stacking interaction between the fluoroquinolone rings (B) offset pattern of the π-π stacking in complex 1.
Interactions between two independent fused-ring moieties: the fluoroquinolone fragment rings and the phen ligands can be found as well in cases of 1 and 2. As it could be expected, these interactions led to interesting molecular packing of the inorganic compounds stabilized by π-stacking interactions ( Fig. S5, S6). The distances from the two centroids of ciprofloxacin (complex 1) or norfloxacin (complex 2) to the phen plane are 3.484 and 3.375 Å, respectively.

Figure S6
Packing diagram of complex IrPNrCu showing (A) π-stacking interaction between the fluoroquinolone rings (B) offset pattern of the π-π stacking in complex IrPNrCu.

Infrared spectroscopies
The FT-IR spectra of the four novel iridium(III)-copper(II) complexes in the far infrared region are shown in in Fig. S10a, b for MIR region; and Fig. 10c shows FT-IR spectra of the starting reagent, S32 Cu(phen)(NO 3 ) 2 : see the Supporting Information). The characteristic (C-H) stretching vibrations generate in the spectral range of 3057-2853 cm -1 the medium peaks in the FT-ATR spectra of discussed complexes.
In the free ligands a strong band near 1720 cm -1 is assigned to the (C=O) stretching vibrations of their carboxylic group (-COOH), 1 which is very weak or not observed in ATR spectra of these complexes.
Besides, in the FT-IR spectra of the novel complexes are observed two characteristic bands attributed to the antisymmetric and symmetric stretching vibrations of (COO -), which can be the marker of the coordination model. The bands with medium intensity around 1630 cm -1 and 1335 cm -1 are assigned to the ν as (COO -) and ν s (COO -) stretching vibrations, respectively (Tab. 3).  respectively. These bands are absent in the spectra of free Ph 2 PCH 2 cfx, Ph 2 PCH 2 nfx, Ph 2 PCH 2 lfx, Ph 2 PCH 2 sfx ligands and phen. Nevertheless, dissolving the obtained complexes in methanol hardly changes their structures, which can be seen in the comparison of the spectra in the solid state with methanol solutions (Fig. S10a,b see the Supporting Information).  Figure S11 a The FT-FIR spectra of complexes 1-4. Figure S11 b The FT-IR spectra of complexes 1-4 in the MIR spectral region.

S35
S36 Figure S11 c The FT-IR spectra of Cu(phen)(NO 3 ) 2 in the MIR and FIR spectral regions.

Figure S12
The X-band EPR spectra of 1 -4 at 77 K together with the spectrum calculated by computer simulation of the experimental spectra with spin Hamiltonian parameters given in the text.

S38
Figure S13 EPR frozen solution spectra (at 77 K) of compounds 1 -4; in DMSO solvent together with the theoretical spectrum calculated using the parameters given in the text. S40 Figure S15 Normalized emission spectra for heteronuclear Ir(III)/Cu(II) complexes, homonuclear Ir(III) complexes and the corresponding phosphine ligands; λex = 340 nm, 298 K.

Magneto-structural studies DC measurements
To fit and interpret the magnetic susceptibility data of examined complexes, first it is necessary to find all possible magnetic pathways. As mentioned in the structural discussion, complexes 3 can be viewed as a monometallic chain in which the neighboring Cu(II) ions are bridged either by carboxylate groups of phosphino-fluoroquinolone ligands or OH-linkers with Cu···Cu separation of 3.470 Å and 4.193 Å, respectively. Considering above the susceptibility data was analysed using a Hamiltonian [eq. 1] for an alternating Ising chain shows in scheme S1: (52) Scheme S1 Alternating Ising chain. , where denotes the z -component of the 2 n-th spin in a linear chain, J 1 and J 2 are 2 The zero-field susceptibility of alternating antiferromagnetic Ising chain is: The best agreement with the experimental magnetic data for 3 was obtained with J1 = -0.  Fig. 7A). The discrepancy factor is 2.09 ×10 -5 (1), 6.93×10 -6 (2) and 3.57×10 -6 (4). These data indicate that a weak exchange interaction between nearest copper atoms in the crystal lattices can exist of antiferromagnetic in 1 and ferromagnetic in 2 and 4 nature. The temperatureindependent paramagnetic term is bigger than usually found. Although the origins of the observed phenomenon are unclear, it was verified by repeated measurements.

S43
New information was obtained from the AC susceptibility measurements. They were performed first at low temperature T = 2.0 K for a set of representative frequencies of the alternating field (f = 1. 1, 11, 111, and 1111 Hz) by ramping the magnetic field from zero to BDC = 1 T with the working amplitude BAC = 0.3 μT.
There was no absorption signal (out-of-phase susceptibility component χ'') at the zero-field owing to a fast magnetic tunneling. With the increasing external field, this component raised, and only for complex 2 passed through a maximum between 0.1 and 0.2 T at the highest frequencies ( Figure S16). This behavior indicates that 2 can exhibit the field induced slow magnetic relaxation. However, the relaxation process for Cu(II) ions is very rare due to the absence of a barrier to spin reversal: the axial zero-field splitting parameter D is undefined. However, some S = 1/2 spin systems such V(IV), S44 low-spin Mn(IV), Ni(I, III), only three example of Cu(II), Os(V), Ir(IV), Fe(III) and Ru(III) complexes show a slow magnetic relaxation (SMR) that is supported by the external magnetic field. (58-62) The presence of a relaxation process in complex 2 can be a result of geometry around Cu(II) ions. Though the D parameter cannot be assigned to mononuclear copper(II) complexes, these are well-known as anisotropic systems showing at least two distinct g z ≠ g x values well seen in the EPR spectra of an axial type. Thus, even in the absence of the zero-field splitting, there exists a magnetic anisotropy. Additionally, the rest complexes not exhibit a relaxation process. This may be due to the presence of higher square-pyramidal geometry distortion in 2 (' = 0.26), which should lead to a greater difference between gx and gy and thus greater anisotropy of g tensor than those for 1, 3 and 4.

Figure S18
Temperature dependence of (a) the in-phase and (b) out-of-phase molar susceptibility of 2.

Characterization of organometallic iridium(iii) complexes
X-Ray quality crystals were obtained by slow evaporation mother liquor. SCXRD measurements for 1 and Electronic absorption spectroscopy was carried out with an UV-Vis spectrophotometer (Agilent Technologies, Cary300 UV-Vis).
Steady state luminescence spectra were acquired with an Edinburgh Instruments FS920 spectrofluorimeter equipped with a R928 phototube detector. The spectroscopic energy E00 was obtained from the crossing of the normalized absorption and emission spectra.
FT-IR spectra of complexes were recorded using a BrukerVertex 70V vacuum spectrometer equipped with a diamond ATR cell with resolution of 2 cm -1 in the middle-infrared (4000-500 cm -1 ) and far-infrared (600-S49 100 cm -1 ) regions at room temperature as solid state and methanolic solutions (c = 0.80%) as well. The spectral data were collected and further elaborated using Bruker OPUS software.
Direct current (DC) magnetic measurements in the temperature range 1.8-300 K (BDC = 0.1 T) and variable -field (0-5 T) (at low temperature) were performed using a Quantum Design SQUID-based MPMSXL-5-type magnetometer. Corrections were based on subtracting the sample -holder signal and contribution χD estimated from the Pascal constants. (69) Variable-temperature (2-7 K) alternating current (AC) magnetic susceptibility measurements under different applied static fields in the range of BDC = 0-1.0 T were carried out with Quantum Design Physical Property Measurement System (PPMS). Magnetic measurements were carried out by crushing the crystals and restraining the sample in order to prevent any displacement due to its magnetic anisotropy.
Electron paramagnetic resonance (EPR) spectra were measured using a Bruker ELEXYS E 500 Spectrometer equipped with NMR teslametr and frequency counter at X-band. The experimental spectra were simulated with use of computer programs (S=1/2) written by Dr. Andrew Ozarowski from NHMFL, University of Florida. The solid compounds dissolved in water and a few drops of DMSO were added to the samples to ensure good glass formation at liquid nitrogen temperature.

S53
Water (1 ml) was poured on the film and Eppendorf tube was homogenized in ultrasonic bath to form light brown or light green liposome suspension. The samples were shaken and heated on Thermomixer comfort for 15 min at 60 ºC.

TEM characterization of liposomes
Liposomes morphology was analyzed by using a FEI™ Tecnai T20 Microscope operated at 200 kV. The size was determined from the enlarged TEM micrographs, using commercially available software ImageJ, counting at least 50 particles in different images. Cells were passaged using a solution containing 0.05% trypsin and 0.5 mM EDTA. All media and other ingredients were purchased from ALAB, Poland.

Cytotoxic activity
Since most of the studied compounds are insoluble in aqueous media, therefore they needed to be pre-

Confocal laser scanning microscopy
The intracellular uptake of 1a was studied in the A549 and DU145 cancer cells according to the previously applied protocol. (4) In brief, before imaging, A549 and Du145 cells were seeded on microscopic slides at a